Glycosylphosphatidylinositols (GPIs)are complex glycolipids that are ubiquitous in eukaryotes. These lipids were discovered covalently linked to cell-surface glycoproteins and recognized to be an important alternative mechanism for anchoring proteins to cell membranes. GPI-anchored proteins appear to be markers of membrane structural domains that are functionally important in intracellular membrane traffic and transmembrane signaling. If GPI transfer to protein is blocked, the protein is not expressed at the cell surface with consequent loss of the relevant cell function. The aim of this continuation proposal is to explore aspects of the assembly and cellular dynamics of GPIs in mammalian cells and protozoa, with the long term goal of acquiring an appreciation of the role of GPIs in cell function. The specific objectives are to study the intracellular transport of GPIs and the covalent modification of proteins by GPI. GPIs are synthesized in the endoplasmic reticulum (ER), and non-protein-linked GPIs are known to be transported to the plasma membrane. We propose that transport occurs by protein-assisted transfer through the cytosol, resulting in the distribution of GPIs to the cytoplasmic face of any receptive cellular membrane. We intend to pursue this hypothesis by studying the sub-cellular distribution and transport of a range of metabolically labeled GPI structures in mammalian cells, by analyses of the transbilayer distribution of GPIs at the plasma membrane (using vesicular stomatitis virus as a topologically correct preparation of plasma membrane) and by re- creating GPI transport in a cell-free system with the aim of isolating transport-relevant cytosolic factors. The overall objective of these studies is to obtain a molecular description of GPI transport in mammalian cells, with the aim of illuminating current notions of intracellular lipid transport and membrane GPIs in signal transduction pathways. GPI attachment to protein occurs via a novel tansamidation reaction in the ER and requires the participation of membrane proteins of the ER. The transamidase activity has been characterized to a limited extent but the putative polypeptide complex corresponding to the enzymatic activity remains to be isolated. We intend to identify the transamidase and other proteins involved in GPI attachment by using cell-free protein translocation-GPI-anchoring systems, in conjunction with chemical and photo crosslinking, and biochemical fractionation. In parallel, we also intend to explore a protein translocation independent assay for transamidase activity that may be used for purification of the activity. Both approaches are expected to uncover candidate proteins involved in GPI anchoring.